Chicken hemogen homolog is involved in the chicken-specific sex-determining mechanism Tomohiro Nakataa, Manabu Ishiguroa, Nana Adumaa, Hiroe Izumib, and Asato Kuroiwaa,b,1 aGraduate School of Life Science, and bLaboratory of Animal Cytogenetics, Department of Biological Sciences, Faculty of Science, Hokkaido University, Hokkaido 060-0810, Japan Edited by Patricia K. Donahoe, Massachusetts General Hospital, Boston, MA, and approved January 18, 2013 (received for review October 29, 2012) Using a comprehensive transcriptome analysis, a Z chromosome- of male chickens. We present evidence that cHEMGN acts as linked chicken homolog of hemogen (cHEMGN)wasidentified and a transcription factor in the nucleus of (pre)Sertoli cells after the showntobespecifically involved in testis differentiation in early sex-determination period and directly or indirectly triggers the ex- chicken embryos. Hemogen [Hemgn in mice, EDAG (erythroid pression of SOX9, suggesting this gene is specifically involved in differentiation-associated gene protein) in humans] was recently chicken sex determination. characterized as a hematopoietic tissue-specific gene encoding a transcription factor that regulates the proliferation and differen- Results and Discussion tiation of hematopoietic cells in mammals. In chicken, cHEMGN was Identification of Chicken Hemogen cDNA and Chromosome Localization. expressed not only in hematopoietic tissues but also in the early High coverage expression profiling (HiCEP) was used to conduct embryonic gonad of male chickens. The male-specific expression a comprehensive transcriptome analysis (10) comparing male was identified in the nucleus of (pre)Sertoli cells after the sex de- and female gonads at day 5.5–6.5 of incubation. A total of 33,962 termination period and before the expression of SOX9 (SRY-box 9). transcripts were identified. The 18 transcripts that were ex- The expression of cHEMGN was induced in ZW embryonic gonads pressed specifically in males or were more than fivefold higher that were masculinized by aromatase inhibitor treatment. ZW em- in males than females were sequenced, and cHEMGN was among bryos overexpressing cHEMGN, generated by infection with retrovi- these transcripts. The full-length coding sequence of cHEMGN rus carrying cHEMGN, showed masculinized gonads. These findings was obtained by RT-PCR and 5′ and 3′ RACE. The full-length suggest that cHEMGN is a transcription factor specifically involved coding sequence was 543 bp, and the predicted amino acid sequence in chicken sex determination. was 180 aa. A bipartite nuclear localization signal and a coiled-coil domain that are present in mouse HEMGN were also conserved bird | gonadal differentiation in cHEMGN (7) (Fig. S1). The amino acid sequence identities be- tween human and chicken, or mouse and chicken, were both 26%. n birds, as in mammals, sex is genetically determined, but males We performed FISH using cHEMGN cDNA clone as probe. fl Iare the homogametic sex (ZZ), and females are heterogametic The uorescence signals were detected in Zq21 in chicken (ZW). The molecular mechanisms determining sex in birds has chromosomes (Fig. S2). This location corresponded with the been a long-standing mystery. In mammals, the sex-determination information of cHEMGN in a chicken genome database (Ensem- gene SRY (sex determining region Y) acts as a transcription factor ble, www.ensembl.org/index.html, last accessed October 20, 2012). to activate SOX9 (SRY-box 9) expression directly by binding to the cHEMGN SOX9 enhancer in pre-Sertoli cells in the undifferentiated gonads Expression Pattern of in Early Embryonic Gonads. Northern cHEMGN of XY embryos (1). SOX9 functions in Sertoli cell differentiation in blot analysis demonstrated that mRNA was more highly expressed in male gonads than female gonads at day 7.5 mammals and other vertebrates (2). A strong candidate for male (Fig. 1A). Quantitative RT-PCR (qRT-PCR) analysis revealed sex determination in chicken is doublesex and mab-3 (Protein that cHEMGN was expressed in the male gonads from day 5.5 MAB-3) related transcription factor 1 (DMRT1), which is on the Z onward, and expression increased dramatically to a peak at day chromosome (3). DMRT1 has been suggested to activate SOX9 8.5 and was then lost before hatching (Fig. 1B). By contrast, fe- indirectly, because there is a time lag between the expression of male gonads exhibited only very low expression throughout em- DMRT1 and SOX9,whicharefirst expressed on day 4.5 and day 6.5 bryogenesis. The expression in male gonads at day 8.5 was more of incubation, respectively (4–6). Therefore, other factors that are fi than 10-fold higher than in female gonads. The cHEMGN pro- likely to be chicken speci c must be in the molecular cascade be- tein was detected in male gonads from day 6.5 onward (Fig. S3). tween DMRT1 and SOX9. Anti-müllerian hormone (AMH) expression in Sertoli cells is one Herein we show that chicken homolog of hemogen (cHEMGN), of the earliest markers of sex differentiation in chicken embryo also in the Z chromosome linkage group, is a transcription factor fi gonads (11). AMH expression was up-regulated between days 5.5 involved in this chicken-speci c molecular cascade. In mice, hem- and 6.5, similarly to cHEMGN expression (Fig. 1C). SOX9 ex- ogen [Hemgn;alsoknownasEDAG (erythroid differentiation- pression was present from day 6.5 and was up-regulated by day associated gene protein) in humans] is a recently characterized fi 8.5 (Fig. 1C). These results are consistent with previous studies hematopoietic tissue-speci c gene encoding a nuclear protein (7). that reported that AMH was expressed in the male gonad before The expression of Hemgn is restricted to the blood islands of the significant SOX9 expression (5, 12). The RT-PCR analysis here yolk sac and the fetal liver during embryogenesis, as well as the revealed that cHEMGN was also expressed before SOX9. BIOLOGY adult spleen and bone marrow (BM) (7). EDAG shows similar expression patterns. EDAG expression is high in the BM cells in DEVELOPMENTAL acute myeloid leukemia, suggesting that EDAG may play a modu- Author contributions: A.K. designed research; T.N., M.I., N.A., and A.K. performed lator role in acute myeloid leukemia (8). Overexpression of Hemgn research; H.I. contributed new reagents/analytic tools; T.N., M.I., N.A., H.I., and A.K. in hematopoietic cells suppresses lymphopoiesis and enhances analyzed data; and T.N. and A.K. wrote the paper. myelopoiesis in transgenic mice, suggesting that Hemgn regulates The authors declare no conflict of interest. the proliferation and differentiation of hematopoietic cells (9). This article is a PNAS Direct Submission. However, the gene is not expressed in the gonads during embryo- 1To whom correspondence should be addressed. E-mail: [email protected]. genesis in mammals. In chicken, cHEMGN was expressed not only This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in hematopoietic tissues but also in the early embryonic gonad 1073/pnas.1218714110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1218714110 PNAS | February 26, 2013 | vol. 110 | no. 9 | 3417–3422 Downloaded by guest on September 28, 2021 Fig. 1. cHEMGN is highly expressed in early embryonic male chicken gonads. (A) Northern blot analysis of cHEMGN in embryonic tissues at day 7.5. cHEMGN mRNA was highly expressed in the male gonad. 18S rRNA was used as a loading control. (B) qRT-PCR of male and female gonads. In male embryos, cHEMGN expression was detected by day 5.5 (just after sex determination) and achieved a peak at day 8.5. The expression was gradually reduced and lost at hatching. Filled square, male; filled circle, female. Data are mean ± SEM; n ≥ 3. (C) RT-PCR of cHEMGN, AMH,andSOX9 in male gonads at day 5.5, 6.5, 7.5, and 8.5. cHEMGN expression was detected before SOX9 expression. GAPDH is the loading control. cHEMGN expression was detected in the gonads of male the SOX9 signal in the gonadal medulla (Fig. 2 G–J). By contrast, embryos using in situ hybridization of whole embryos (WISH) the cHEMGN signal did not colocalize with that of CVH (Fig. 2 K– (Fig. 2A) and in the gonadal medulla using frozen sections of N). The cHEMGN protein was observed in the nucleus of male embryos (Fig. 2 C and D). No signals were detected in the gonads gonadal cells by immunohistochemistry (IHC) (Fig. 2 O–R). These of female embryos (Fig. 2 B, E,andF). To identify the cells that results suggested that cHEMGN was a nuclear protein expressed in expressed cHEMGN, double-label in situ hybridization was con- Sertoli cells. ducted using SOX9 and chicken vasa homolog [CVH, also known as DDX4 (DEAD box polypeptide 4)] as markers for Sertoli cells and Expression Pattern of cHEMGN in Hematopoietic Tissues. The ex- germ cells (13), respectively. The cHEMGN signal colocalized with pression of cHEMGN in chicken hematopoietic tissues was Fig. 2. cHEMGN is expressed in the nucleus of Sertoli cells within the medulla of the male gonads. (A and B) WISH of male and female embryos at day 7.5. cHEMGN was expressed throughout the male gonads. The dashed lines indicate the gonads. (Scale bar, 300 μm.) (C–F) cHEMGN in situ hybridization in frozen sections of male and female gonads at day 7.5. cHEMGN expression was localized to the medulla of the male gonad. The negative control used a sense probe for hybridization. (Scale bar, 100 μm.) (G–N) Dual-labeled in situ hybridization of frozen sections of male and female gonads at day 8.5. cHEMGN expression colocalized with SOX9 expression in Sertoli cells. (Scale bars, 100 μm.) (O–Q) IHC of day-8.5 male gonads using a cHEMGN antibody. cHEMGN was expressed in the nucleus of Sertoli cells in the medulla of the male gonad.